Fluorescence Imaging Analysis Of The Microenvironment In Brain Nerve Cells

Depression with high morbidity and mortality rate seriously impairs people's physical and mental health.The neurotrophic hypothesis maintains that depression is associated with the decreased expression and functional degeneration of brain derived neurotrophic factor(BDNF)assembled from it precursor protein in Golgi apparatus.These changes are closely related to the variation in the microenvironment of Golgi apparatus.Thus,developing nondestructive,in situ and instantaneous method to detect microenvironment of Golgi apparatus in live cells and brain tissues can contribute to accurate diagnosis and prognosis of depression.Herein,we designed two Golgi apparatus-targetable fluorescent probe for imaging the variations in polarity and pH of Golgi apparatus.We carried out the following two parts of our research work.1.In situ fluorescence imaging of Golgi polarity in living brain of depression mice.Golgi-P was made of merocyanine as an electron-donating group and benzoyl difluoroboron as an electron-withdrawing group.They formed the polar-sensitive conjugated structure which resulted to excitation and emission wavelengths in the nearinfrared region.The linking of L-cysteine to the nitrogen atom of merocyanine allowed accurate positioning of the Golgi apparatus.Upon excited with 700 nm,the fluorescence intensity of Golgi-P showed obvious polarity dependence accompanying the shift of maximum emission wavelength.The imaging results demonstrated that the polarity of Golgi apparatus in glutamate-stimulated PC12 cells significantly increased compared to normal cells.Furthermore,by virtue of Golgi-P,we discovered distinctly higher polarity in the brain tissues of depressed mice than normal mice for the first time.Meanwhile,the BDNF kit test verified the decreasing synthesis of BDNF in the brain tissues of depressed mice,which might be a major cause of elevated polarity.2.In situ fluorescence imaging of Golgi pH in living brain of depressive mice.Golgi-pH consisted of Rhodamine B as a fluorophore and L-cysteine as a positioning group for the Golgi apparatus.We used the lactam to control the fluorescence intensity to monitor the real-time changes in pH of the Golgi apparatus.If the pH of the Golgi apparatus rises,the fluorescence weakens or even disappears.On the contrary,the fluorescence gradually increases.